Existing therapies, particularly against malaria, are under constant pressure from acquired parasite drug resistance requiring a continuing search for new treatments. The peculiar proliferative cycles of Apicomplexa parasites differ substantially from the hosts they inhabit and should offer fertile ground to supply an active pipeline of new targets. To fulfill this promise, we need a better understanding of the unique structural and molecular features of parasite cell division. Apicomplexan proliferation has adapted to different host cells using chromosome replication cycles that can vary in the scale of nuclear reduplication from a few to hundreds of nuclei produced per day, which is unparalleled cell cycle flexibility. How fidelity is preserved through variable rounds of chromosome replication is a major mystery of Apicomplexa biology as many known regulators of DNA replication in multicellular eukaryotes are missing in these parasites. Further, the basic checkpoint mechanisms that regulate the cell cycle transitions are also poorly understood. It is not known what controls G1 to S phase commitment, S phase progression, chromosome segregation or what controls allow the parasite to forgo budding in one type of chromosome cycle but not in another during the processes of schizogony and endopolygeny. In this application we will investigate several Toxoplasma mutants that carry lethal point mutations in proteins essential for proper chromosome replication and segregation. When shifted to high temperature these mutants all suffer very similar disruptions in chromosome replication. In Aim 1, we will define the molecular function of a novel RING protein (ECR1, essential for chromosome replication 1) in regulating chromosome replication and segregation. We will determine whether ECR1 is a divergent E3 ligase and compare the molecular features of this mechanism in controlling DNA replication to the known chromosome licensing factor Topo-II. We will also investigate an alternate role for ECR1 in regulating the tachyzoite cell cycle. ECR1 forms a complex with a Toxoplasma ortholog of human cyclin-dependent kinase 2 (TgCDK2). The ECR1/TgCDK2 complex appears in the centrocone and then leaves this compartment to become exclusively nuclear during S phase. We will characterize the molecular basis for this interaction and determine whether this partnership is required for ECR1 function. We will also determine the basic features of the TgCDK2 mechanism including whether it requires a cyclin for function and identify the protein substrates of TgCDK2 in order to understand how this kinase mechanism regulates the tachyzoite S phase progression. In Aim 2, we will investigate two chemical mutants harboring two other defective ECR factors (ECR2 and 3) that also cause uncontrolled DNA synthesis when mutant parasites are grown at high temperature. ECR2 and ECR3 are unknown proteins conserved only within Apicomplexa genetic lineages. The discovery of apicomplexan-specific factors essential for chromosome replication supports our central hypothesis that these ancient parasites have evolved unique molecular mechanisms to regulate asexual stage proliferation.